Anti-Leishmania major activity of Calotropis procera extract by increasing ROS production and upregulating TNF-α, IFN-γ and iNOS mRNA expression under in vitro conditions

Background Leishmaniasis, caused by protozoan parasites of the genus Leishmania, is a neglected tropical disease with 700,000 to 1,000,000 global new cases annually. Adverse effects associated with expense, long-term treatment and drug resistance have made conventional therapies unfavorable, encouraging the search for alternative drugs based on plant products. In this study, the effect of Calotropis procera (Asclepiadaceae) extract against viability of promastigotes and amastigotes of Leishmania major was evaluated in vitro. Methods The extract from the leaves of C. procera seedlings was prepared using a methanol maceration method. The colorimetric cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth-inhibitory effect of the extract on promastigotes. The level of reactive oxygen species (ROS) in promastigote cultures was determined after treatment with the extract using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method and compared with untreated cultures (control). After exposure to the extract the expression levels of tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) genes were determined and compared to control in peripheral blood mononuclear cells (PBMCs) infected with L. major. Results Based on the MTT assay, the C. procera extract significantly reduced the proliferation of L. major promastigotes with IC50 values of 377.28 and 222.44 μg/mL for 24 and 72 h, respectively (p < 0.01). After treatment with 222.44 and 377.28 μg/mL of C. procera extract, ROS production in L. major promastigote cultures increased 1.2- to 1.65-fold and 2- to 4-fold compared to the control, respectively (p < 0.05). C. procera extract induced significant increases in gene expression of TNF-α (2.76–14.83 fold), IFN-γ (25.63–threefold) and iNOS (16.32–3.97 fold) in infected PBMCs compared to control (p < 0.01). Conclusions On the basis of its anti-leishmanial activity, C. procera can be considered as a promising new plant source for the potential treatment of leishmaniasis.


Background
Leishmaniasis is a neglected obligatory intracellular tropical disease, caused by different species of Leishmania parasites, that is prevalent in many parts of the world [1].Leishmaniasis is transmitted by the bites of infected female sand flies [2].These parasitic protozoans have two distinct stages in their life cycle: promastigotes (extracellular flagellated promastigotes in the gut of the female sand fly vector which can be injected into the host dermis by vector bite), and amastigotes (transformation of promastigotes into intracellular amastigotes after internalization by host phagocytotic cells through phagocytosis) [3,4].At the vector bite site, the parasite in promastigote form attacks host phagocytotic cells (inflammatory monocytes, macrophages and neutrophils) and then transform into intracellular amastigotes [4,5].Amastigotes are able to proliferate within monocytes/macrophages and transmit the infection to other macrophages, neutrophils, monocytes, some dendritic cells and fibroblasts [4,6,7].
The defense reactions of host cells against Leishmania infection are based on the co-ordination of two host immune systems, innate immunity (complementmediated lysis) and adaptive immunity (Th1-mediated response) [4,5].The expression of activating cytokines such as IFN-γ and TNF-α is essential for parasite proliferation control [8].When activated by cytokines, host cells can suppress the infection by killing intracellular parasites [9].The production of reactive oxygen species (ROS) and Nitric Oxide (NO expressed by inducible nitric oxide synthase (iNOS) gene) represent as two major effective leishmanicidal molecules for exclusion of intracellular parasites without damaging the host cell [4,5].
Treatment of leishmaniasis has always been challenging.The absence of effective immunizations and/or emergence of treatment resistance have all contributed to the rise in prevalence of this disease [10].Pentavalent antimonials, amphotericin B, paromomycin and pentamidine are the most regularly used drugs for leishmaniasis therapy.However, they have significant side effects, require high dosages for extended periods of time, and are supplied parenterally [11].An effective and economical new treatment approach would be advantageous in overcoming the difficulties induced by leishmaniasis chemotherapy.In the treatment of parasitic infections, phytotherapy has recently attracted attention as a viable alternative to chemotherapy [12].In this regard, plant studies have been expanded to discover new secondary metabolites with increased bioactivity and fewer side effects [12,13].
The mechanisms and pathways that stimulate leishmania-infected macrophages are of special interest since they hold potential for the development of new treatment and prevention strategies.In the present research, we sought to evaluate the effect of C. procera extract on PBMCs infected with L. major and the expression levels of INF-γ, TNF-α and iNOS genes.It was hypothesized that treatment of L. major promastigotes and amastigotes with C. procera extract would induce ROS production and upregulation of INF-γ, TNF-α and iNOS genes which could be effective for parasite control.

Preparation of plant extract
The seeds of C. procera were provided and cultivated in the greenhouse (Fig. 1) by Zarringiah Co., West Azerbaijan Province, Urmia, Iran.The growing seedlings were authenticated by a taxonomist.Voucher specimens (voucher numbers: CP/1397 433) were deposited in the Zarringiah Co. Herbarium, Urmia, Iran.The leaves of 6 weeks seedlings were carefully harvested for extraction (Fig. 1).After washing and shade drying at room temperature, the samples (500 mg) were powdered and extracted by 10 ml 80% methanol (Merck, Darmstadt, Germany) maceration method and shaking incubation at 25 ± 2 C for 72 h.The extract was paper filtered and the residue re-macerated for the second (80% methanol, 48 h) and third (80% methanol, 24 h) time.Finally, the solvent was evaporated in a vacuum rotary evaporator (Rotary Evaporator N-1110, Eyela, Tokyo, Japan).The concentrated residue was frozen at − 20 C. The dried powders were dissolved in phosphate-buffered saline (PBS, Cl 2 H 3 K 2 Na 3 O 8 P 2 , 1X, pH 7.4, Gibco, Paisley, UK) and diluted to prepare test concentrations of extract.

Cultivation of Leishmania major parasite
The Iranian standard reference strain of L. major promastigotes (MRHO/IR/75/ER) was provided by the Department of Medical Parasitology and Mycology, Urmia University of Medical Sciences, Urmia, Iran.The promastigotes were cultured in RPMI-1640 culture medium (+ HEPES and L-glutamine, Gibco, Paisley, UK) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, PAN Biotech, Aidenbach, Germany) and antibiotics (100 units/ml penicillin, and 100 μg/ml streptomycin, Sigma-Aldrich, St. Louis, Missouri, USA).The cultures were placed in an incubator shaker (120 rpm) at 25 ± 1 C and grown until reaching the stationary growth phase.
The logarithmic regression analysis of dose-response curve was used for calculation of 50% inhibitory concentration of extract (IC 50 ) and 50% cytotoxic concentration of extract (CC 50 ) using GraphPad Prism 5.0.4 software (GraphPad Software, San Diego, California, USA).

Cultivation of PBMCs and infection with L. major promastigotes
Peripheral blood mononuclear cells were isolated from healthy heparinized blood as described by Srivastava et al. [28] and cultured in 6-well plates (10 5 cells/well) containing RPMI 1640 medium (+ HEPES and L-glutamine, Gibco, Paisley, UK, 10% FBS (PAN Biotech, Aidenbach, Germany), 100 U/ml penicillin-100 µg/mL streptomycin (1% P/S, Sigma-Aldrich, Missouri, USA) as antibiotics) at 37 C -5% CO 2 .The adherent cells were infected with L. major promastigotes at stationary growth phase (10:1 parasites/cell) for 4 h and washed three times with PBS (Gibco, Paisley, UK) to remove free parasites.After stabilization of infected cells (amastigote-containing cells) the treatments were performed for 24, 48 and To determine the cytotoxicity effect of C. procera leaf extract on PBMCs, the concentration of extract required to reduce uninfected PBMC growth by 50% after 24-72 h was calculated using the MTT assay.

IFN-γ, TNF-α and iNOS mRNA determination by real-time PCR
Total RNA from infected PBMCs (treated or untreated with extract) was extracted using SinaClon RNXplus kit (SinaClon, Tehran, Iran).Synthesis of cDNA was performed with 1 μg of total RNA using the AccuPower ® CycleScript RT PreMix Kit (Bioneer, Daejeon, South Korea) according to the manufacturer's instructions.The specific primers targeting the genes were designed as listed in Table 1, and manufactured (Nedaye Fan Co, Tehran, Iran).The Real-time RT-PCR assays were performed by SYBR Green detection (SYBR Green qPCR Master Mix, Thermo Scientific/Fermentas, Vilnius, Lithuania) and the relative quantification (2 −ΔΔCT method) was applied, using the homo-sapiens β-actin gene as the reference control.Real-time RT-PCR reactions were conducted using three-step real-time MicPCR (Bio Molecular system, Upper Coomera, Queensland, Australia) in 20 μL total volume containing 10 μL SYBR Green Master Mix, 2 μL of 1:20 diluted cDNA (50 ng), 0.5 µL of each primer (10 µM), and 7 µL nuclease-free water.The realtime PCR temperature program consisted of a hold at 95˚C for 10 min followed by 40 thermal cycles of 95˚C for 15 s, primer annealing temperature (Table 1) for 20 s, and 72 C for 30 s.

Statistical analysis
Values were expressed as the mean of triplicate samples ± standard deviation (SD).The results were analyzed statistically by one-way ANOVA test followed by Duncan's multiple range tests (p < 0.05).

In vitro leishmanicidal activity of C. procera against L. major promastigotes
The effect of C. procera leaf extract on L. major promastigotes was monitored after 24 and 72 h of treatment.The extract showed a dose-dependent reduction in promastigote proliferation (Fig. 2), with 50% growth inhibition of the promastigotes at 377.28 μg/mL extract after 24 h and 222.44 μg/mL extract after 72 h of treatment.In order to ensure the selectivity of C. procera leaf extract to act only against intracellular amastigotes, the cytotoxicity against PBMCs was investigated.No cytotoxicity was observed at the concentrations analyzed (selectivity index (SI) higher than 4, Table 2).

C. procera extract increased ROS production
Overproduction of ROS in mitochondria is one of the important defense weapons of the cell against pathological and physiological threats that lead to oxidative stress.We determined the ROS production in L. major promastigotes using fluorescent H2DCFDA detection by flow cytometry.Promastigotes treated with 222.44 and 377.28 µg/mL C. procera extract significantly enhanced ROS production by 1.65 and 4 times (p < 0.001), respectively, compared to controls (Fig. 3).

C. procera extract increased expression of IFN-γ and TNF-α transcripts
As shown in Fig. 4, in L. major-infected PBMCs, IFN-γ and TNF-α mRNA expression increased significantly during treatment with C. procera, depending on exposure time (p < 0.01).The highest expression of both IFN-γ and TNF-α genes was detected at 48 h treatment with 377.28 µg/mL C. procera extract, compared to control expression levels (p < 0.001).In the presence of 222.44 µg/mL C. procera extract, cytokine expression in L. major-infected PBMCs significantly increased with increasing exposure time (p < 0.001), such that 72 h treatment induced higher levels of TNF-α and IFN-γ in comparison with control, respectively (Fig. 4).

Discussion
The current study represents the first report of C. procera and its impact on the expression of relevant genes in L. major-infected PBMCs.Our findings demonstrated that C. procera could induce ROS generation in L. major promastigotes; increase expression of IFN-γ and TNFα cytokine genes, together with nitric oxide synthase expression, in L. major-infected PBMCs.Considered together, this indicated an inhibitory effect on the proliferation of L. major promastigotes.C. procera is known to possess antioxidant, antipyretic, antifungal, antimicrobial, analgesic, anti-inflammatory and antinociceptive properties which have been attributed to its phytochemical composition [18,[22][23][24].In the current study, the anti-leishmanial effects of C. procera on L. major promastigotes were evaluated by MTT assay.Our findings showed that C. procera had a dosedependent cytotoxic effect against L. major promastigotes, as also reported against L. tropica species [26].The IC 50 value obtained from leaf extract of C. procera against L. major promastigotes in the present analysis was 377.28 μg/mL at 24-h treatment and 222.46 μg/m at 72-h treatment which was higher than the value reported (66.8 μg/mL -72 h) in Al Nasr' study [29].It has been clear that the biosynthesis and accumulation of secondary metabolites in plants are influenced by genetic and environmental factors [30].Therefore, it is not far from expected that the IC 50 s of the same plant but grown in different environmental conditions are different.C. procera comprises secondary metabolites such as phenolic compounds, flavonoids, cardiac glycosides, terpenoids,  saponins and sterols [17].The anti-leishmanial effects of these phytochemicals against Leishmania spp.have been demonstrated previously [31].Therefore, these phytochemicals may have been responsible for the antileishmanial activity of C. procera in the current study.Phenolic acids such as gallic acid and ellagic acid [32], and flavonoids, for example, rutin [33] have shown growth-inhibitory effect against promastigotes and amastigotes of L. major and L. donovani, respectively.Based our results, in a dose-time-dependent manner, L. major promastigote proliferation was inhibited after treatment with C. procera extract as a consequence of increased ROS production.It can be stated that after mitochondrial dysfunction and as a result leakage in the electron transport chain, the level of ROS in Leishmania promastigotes will exceed its basal level [34].Phytochemicals possessing anti-leishmanial activity may therefore have been able to increase ROS levels, resulting in oxidative stress produced by ROS, thereby causing cell death [31].Similarly, other studies have pointed out the important role of herbal products in inducing excessive production of ROS and subsequent cell death of Leishmania spp.For example, Dehydroabietic acid isolated from Pinus elliottii in the Gonçalves, et al. study [35] and total phenolic fraction from extra virgin olive oil in the Karampetsou, et al. study [34], promoted cellular ROS production in L. amazonensis and L. major parasites, respectively.
In the current study, 48 h treatment with 377.28 µg/ mL C. procera induced IFN-γ and TNF-α expression and increased iNOS gene expression in L. majorinfected macrophages, leading to elimination of the parasites.To success of host's defense mechanisms against Leishmania parasite, IFN-γ and TNF-αas proinflammatory cytokines play crucial role [36].Evidently, FN-γ and TNF-α have synergistic effects in killing of Leishmania major by stimulating macrophages to increase ROS and reactive nitrogen species (RNS) production [4,5,36].As a result of increased iNOS gene expression, host cells produce NO via activity of the iNOS enzyme [37].NO is known to be a major leishmanicidal agent, such that deficiency or inhibition of NO production leads to parasite resistance or survival, respectively [37].It has been demonstrated that injection of L-NG-monomethyl arginine (L-NMMA), as a NO inhibitor, into the lesions in L. major-infected CBA mice caused disease exacerbation by 10(4)-fold increasing in the number of parasites [38].
According to these findings, increased iNOS expression could represent an effective mechanism for C. procera to control Leishmania infection; supported by the RT-PCR results in the current study.It is evident that antimonials, amphotericin B and other anti-leishmanial agents have been shown to combat parasites by increasing production of ROS and NO [39,40].Similarly, the increased release of IFN-γ and TNF-α by C. procera leaf extract could therefore represent an underlying immune mechanism to stimulate iNOS expression and thus NO production.Considering the safety,

Conclusions
In the current study, C. procera leaves extract exerted significant anti-leishmanial activity against L. major promastigotes and amastigotes.This was likely mediated by effective concentrations of C. procera extract increasing the levels of ROS in promastigotes, and increasing the expression levels of IFN-γ, TNFα and iNOS genes in PBMCs containing amastigotes.Considering that despite the high efficacy rate, the presence of severe side effects, toxicity, some drug resistance and the high cost of chemical anti-leishmanial drugs encourage efforts to find effective, safe and

Fig. 2
Fig.2MTT assay of L. major promastigotes viability after C. procera leaf extract treatments.Data are expressed as mean of % cell viability ± standard deviation.Significant statistical differences in relation to control are indicated as (*), (**), and (***) at the 0.05, 0.01 and 0.001 levels, respectively

Table 2
In vitro activity of C. procera against L. major promastigotes and its cytotoxicity for PBMCs